The phytochromes comprise a family of biliprotein photoreceptors that enable plants to adapt to their prevailing light environment (Kendrick and Kronenberg (1994) Kendrick, Pp. 828 in Photomorphogenesis in Plants, Dordrecht, The Netherlands: Kluwer Academic Publishers). Phytochromes possess the ability to efficiently photointerconvert between red light absorbing Pr and far red light absorbing Pfr forms, a property conferred by covalent association of a linear tetrapyrrole (bilin or phytobilin) with a large apoprotein. Phytochromes from cyanobacteria, to green algae and higher plants consist of a well conserved N-terminal polypeptide, roughly 390–600 amino acids in length (see, e.g. U.S. Pat. No. 6,046,014), to which the phytobilin prosthetic group, e.g., phytochromobilin (PΦB) or phycocyanobilin (PCB) is bound.
Phytobilins are linear tetrapyrrole molecules synthesized by plants, algae, and cyanobacteria that function as the direct precursors of the chromophores of the light-harvesting phycobiliproteins and of the photoreceptor phytochrome (Beale (1993) Chem. Rev. 93: 785–802; Hughes and Lamparter (1999) Plant Physiol. 121: 1059–1068). The pathways of phytobilin biosynthesis have been elucidated by biochemical fractionation of plant and algal extracts, by overcoming a blocked step with exogenous putative intermediates, and by analysis of linear tetrapyrrole-deficient mutants (Beale and Cornejo (1991) J. Biol. Chem. 266: 22328–22332; Beale and Cornejo (1991) J. Biol. Chem. 266: 22333–22340; Beale and Cornejo (1991) J. Biol. Chem. 266: 22341–22345; Terry et al. (1993) Arch. Biochem. Biophys. 306: 1–15). These studies indicate that the biosynthesis of phytobilins shares common intermediates with heme and chlorophyll biosynthetic pathways to the level of protoporphyrin IX, at which point the latter two pathways diverge by metalation with iron or magnesium (Beale (1993) Chem. Rev. 93: 785–802). Phytobilins are derived from heme, which is converted to biliverdin IXa (BV), the first committed intermediate in their biosynthesis. In red algae, cyanobacteria, and plants, this interconversion is accomplished by ferredoxin-dependent heme oxygenases that are related in sequence to the mammalian heme oxygenase (Cornejo et al. (1998) Plant J. 15: 99–107.; Davis et al. (1999) Proc. Natl. Acad. Sci., USA, 96: 6541–6546; Muramoto et al. (1999) Plant Cell 11: 335–347). Although they catalyze the same reaction, mammalian heme oxygenases use an NADPH-dependent cytochrome P450 reductase to generate reducing power for heme catabolism (Maines (1988) FASEB J. 2: 2557–2568).
The metabolic fate of BV differs in mammals, cyanobacteria, and plants, with BV being metabolized by different reductases with unique double-bond specificities (FIG. 1). Mammalian biliverdin IXa reductase (BVR), an NAD(P)H-dependent enzyme that catalyzes the two-electron reduction of BV at the C10 methine bridge to produce bilirubin IXa (BR), was the first of these enzymes to be discovered (Maines and Trakshel (1993) Arch. Biochem. Biophys. 300: 320–326). A similar enzyme, encoded by the gene bvdR, was identified in cyanobacteria (Schluchter and Glazer (1997) J. Biol. Chem. 272: 13562–13569). Cyanobacteria and red algae also possess novel ferredoxin-dependent bilin reductases for the synthesis of the linear tetrapyrrole precursors of their phycobiliprotein light-harvesting antennae complexes (Beale and Cornejo (1991) J. Biol. Chem. 266: 22328–22332; Beale and Cornejo (1991) J. Biol. Chem. 266: 22333–22340; Beale and Cornejo (1991) J. Biol. Chem. 266: 22341–22345; Cornejo et al. (1998) Plant J. 15: 99–107). Primarily on the basis of studies with the red alga Cyanidium caldarium, these investigators proposed that the biosynthesis of the two major phycobiliprotein chromophore precursors, phycoerythrobilin (PEB) and phycocyanobilin (PCB), utilized two ferredoxin-dependent bilin reductases and several double-bond isomerases. The first bilin reductase catalyzes the two-electron reduction of BV at the C15 methine bridge to produce the BR isomer 15,16-dihydrobiliverdin (DHBV), whereas the second bilin reductase catalyzes the conversion of 15,16-DHBV to 3Z-PEB, a formal two-electron reduction of the C2 and C3 diene system. In C. caldarium, an additional enzyme mediates the isomerization of 3Z-PEB to 3Z-PCB, both of which appear to be isomerized to their corresponding 3E isomers before assembly with the nascent phycobiliprotein apoproteins (Beale and Cornejo (1991) J. Biol. Chem. 266: 22328–22332; Beale and Cornejo (1991) J. Biol. Chem. 266: 22333–22340; Beale and Cornejo (1991) J. Biol. Chem. 266: 22341–22345).
More recent studies lend support for a similar pathway of PCB and PEB synthesis in cyanobacteria (Cornejo and Beale (1997) Photosynth. Res. 51: 223–230). In contrast with mammals and phycobiliprotein-containing organisms, plants and green algae reduce BV to 3Z-PFB by the ferredoxin-independent enzyme PΦB synthase, which targets the 2,3,31,32-diene system for reduction (Terry et al. (1995) J. Biol. Chem. 270: 11111–11118; Woo et al. (1997) J. Biol. Chem. 272: 25700–25705). In plants, 3Z-PFB is isomerized to its 3E isomer, which appears to be the immediate precursor of the phytochrome chromophore (Ibid). The green alga Mesotaenium caldariorum possesses a second bilin reductase activity that catalyzes the reduction of the 18-vinyl group of PFB to produce 3Z-PCB (Wu et al. (1997) J. Biol. Chem. 272: 25700–25705). These investigations also revealed that 3E-PCB is the natural phytochrome chromophore precursor in this organism.
Despite the extensive biochemical analysis of the phytobilin biosynthetic pathways in plants, algae, and cyanobacteria, the low levels of bilin reductase expression have previously hindered efforts to clone these enzymes.